Equilibrative nucleoside transporters (ENTs) are the predominant membrane transporters in humans mediating the uptake and efflux of adenosine, which is an important inter-cellular signaling molecule mediating a wide range of effects from neuro- and cardioprotection to pain perception, susceptibility to seizures and sleep cycle. Despite their immense significance in human physiology and pathophysiology, little is known about the mechanism by which ENTs recognize and translocate their substrates across hydrophobic lipid bilayers. In this proposal we focus on the crystallization of ENT homologues, a critical step towards determining the crystal structure of these molecules. We have already identified ENT homologues with superb structural stability in detergent and established expression and purification protocols for them that routinely yield milligram quantities of stable homogeneous proteins. We will further optimize the protein constructs, detergents, ligands and lipid supplementation to achieve transporters crystallization. An important concern is that membrane transporters are dynamic proteins by nature, and that in solution they may distribute over multiple state hindering crystallization. To circumvent this problem, we will systematically mutate 27 highly conserved amino acids within hENT1, which is one of the crystallization candidates, and implement a functional screen in yeast in order to identify the loss-of-function mutants, that are highly expressed, stable in detergent and capable of tight substrate binding but not transport. We hypothesize that such mutants will be deficient in transport because they are unable to undergo some or all of the essential conformation transitions and are, thus conformationally constrained. We will evaluate whether such mutants would provide distinct benefits in the crystallization process. The success of this project will have a major impact on the studies of ENTs and possibly on the larger field of the structural biology of dynamic membrane proteins such as channels and transporters. PUBLIC HEALTH RELEVANCE: Adenosine is an important signaling molecule in humans, regulating a wide range of effects such as vasodilatation, thrombus formation, pain perception, sleep cycle, and body temperature. Specialized proteins in the cellular membranes, equilibrative nucleoside transporters control the levels of adenosine available for signaling. They are the targets of many drugs such as dipyridamole, which is widely used to prevent secondary strokes. Here we propose to crystallize these molecules, an essential step toward determination of their atomic structure, which in itself is essential to understand the transporters mechanism and to increase our capacity to pharmacologically control their function.